Abstract: ob＜x＞jective To determine the structure and characteristics of intestinal flora in EV71 hand-foot-mouth disease. Methods From January 2020 to December 2021, fecal samples of EV71 hand-foot-mouth disease and healthy children diagnosed in Suqian Hospital Affiliated to Xuzhou Medical University were collected. After extracting DNA from feces, 16S rDNA amplicon sequencing was performed, and the obtained biological information was classified, quantified and compared. α-diversity was used to analyze the differences in abundance and diversity between the two groups. β-diversity was used to analyze the differences in bacterial community structure between the two groups. LDA analysis and LEfSe analysis were used to compare the significantly different genera between the two groups. Results A total of 80 fecal samples were collected from children with EV71 hand -foot-mouth disease (EV71 group, 45 cases) and healthy children (control group, 35 cases). Compared with the control group, there were no significant changes in the diversity (shannon index, P>0.05) and abundance (ace index, P>0.05) of intestinal flora in the EV71 group . However, the structure of intestinal flora in EV71 group was significantly changed (weighted_unifrac, P<0.05), which was mainly manifested in the increase of the abundance of Neisseria ( P<0.05) and Klebsiella ( P<0.01), while the abundance of Romboutsia ( P<0.01), Dialister (P<0.01) and Clostridium ( P<0.05) were decreased . Conclusion The intestinal flora of EV71 hand-foot-mouth disease is disordered, which is mainly characterized by the decrease of butyrate producing bacteria and the increase of opportunistic pathogenic bacteria. The results provide some theoretical basis for the pathogenesis and microecological treatment of EV71 hand-foot-mouth disease in the later stage.